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There is a need for more objective and quantitative tools to replace microscopy in malaria diagnosis. Emphasis has recently been placed on alternative methods such as immunochromatography-based rapid tests. However, these tests provide only qualitative results. Two bio-molecules, parasite lactate dehydrogenase (pLDH) and histidine-rich proteins (HRPs), that are released by the intra-erythrocytic stages of the parasite offer certain specific characteristics that could potentially improve malaria diagnosis. In this paper, we describe a protocol for a unified sandwich ELISA that allows for the separate but concurrent measurement of pLDH and HRP biomolecules in aliquots taken from the same samples. Freshly drawn blood from a healthy unexposed adult male was used to serially dilute in vitro cultivated and synchronized ring stage Plasmodium falciparum parasites. Commercially available ELISA formats were modified to allow for the measurement of pLDH and HRP from aliquots of the same samples. The pLDH and HRP levels in the samples spiked with known numbers of infected red blood cells (iRBCs) were measured, and the values were used to generate standard graphs. The standard graphs were used to estimate the numbers of iRBCs in test samples. Serially diluted recombinant proteins were similarly used to generate a calibration curve, allowing for the expression of test results in nanograms of their respective recombinant protein. Levels of pLDH and HRPs were determined by using 1) P. falciparum culture material (cells and medium) 2) P. falciparum infected human blood (N = 6) samples, and 3) plasma from P. falciparum–infected patient (N = 22) samples. The parasite density of all culture and infected patient samples was also estimated by microscopy. Both pLDH and HRP levels correlated positively with the parasite density assessed by microscopy: Pearson correlation coefficient pLDH (r = 0.754, P < 0.0001, 95% CI: 0.47–0.89); HRP (r = 0.552, P < 0.007, 95% CI: 0.16–0.79). The HRPs seem to be released in larger quantities than pLDH (in a ratio of ~1 pLDH:~6 HRP), making the detection of HRP in culture material, blood, and plasma easier. The modified ELISA assay with quantitative measurement of pLDH and HRPs may provide a valuable tool for malaria research and patient management.
Received March 28, 2008. Accepted for publication October 22, 2008.
Acknowledgments: The authors thank Dr. B. Ogutu and staff at the USAMRU-K Kombewa clinic for microscopy support and Dr. D. Sullivan of Johns Hopkins University, Baltimore, MD, for useful comments and provision of the HRP-2 recombinant proteins. GRR is a consultant at Cellabs Pty of Sydney, Australia. Our thanks are extended to Lucy Thuita for statistical analysis, Dr. M. Makler of Flow Inc., Portland, OR, and Dr. Anthony Smithyman of Cellabs Pty, Sydney, Australia, for critically reviewing the manuscript.
Financial support: This work was done with funding from the US Agency for International Development and the permission of the Director, Kenya Medical Research Institute, Nairobi, Kenya.
Disclaimer: The views expressed are those of the authors and do not purport to reflect the positions of the US Department of Defense, the USAID, or the vendors of the commercial kits.
* Address correspondence to G-Halli Rajasekariah, Biofirm Pty, 19 Burraneer Avenue, St. Ives, NSW 2075 Australia. E-mails: raj{at}cellabs.com.au and rajasekariah{at}optushome.com.au
Authors’ addresses: Samuel K. Martin, Unit 64109, APO AE 09831-4109. G-Halli Rajasekariah, Biofirm Pty, 19 Buccaneer Avenue, St Ives, NSW 2075, Australia. George Awinda, John Waitumbi, and Carolyne Kifude, Unit 64109, APO AE 09831-4109.
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